Previously, we demonstrated the existence of a set of eight genes that are essential for the secretion of pertussis toxin (PT) from B. pertussis. These genes, termed ptl genes, encode transport proteins that share homology with a family of proteins involved in the transport of both DNA and proteins across bacterial membranes. We now have evidence for a ninth secretion gene. This small gene, ptlI, is predicted to encode a protein of 6.8 kDa and is located between ptlD and ptlE. PtlI shares homology with VirB7 which is required for transport of T-DNA across the membranes of the plant pathogen Agrobacterium tumefaciens. Deletion of the ptlI region from B. pertussis has no discernable effect of PtlE but resulted in loss of detectable PtlF, suggesting that PtlI may interact with and stabilize PtlF. Using immunoprecipitation techniques, we demonstrated a direct interaction between PtlI and PtlF. These results are the first evidence for interactions between Ptl proteins. Work is continuing to further elucidate the structure of a Ptl transport apparatus. In addition to the work on elucidation of interactions between Ptl proteins, we are also investigating the roles of individual Ptl proteins in the transport process. Two Ptl proteins, PtlC and PtlH, have the interesting characteristic that they each contain a nucleotide binding motif. We have demonstrated that alteration of this region in PtlH using site-specific mutagenesis destroys the ability of B. pertussis to secrete PT. Moreover, this mutation has a dominant negative phenotype suggesting that PtlH either oligomerizes or forms complexes with other proteins. We are currently investigating the importance of the nucleotide-binding region of PtlC. We hope to purify both PtlH and PtlC and determine whether they bind nucleotides and, if so, whether they contain enzymatic activity such as a hydrolase or kinase activity. This work increases our knowledge of the secretion of PT and may aid in the construction of strains of B. pertussis which efficiently produce and secrete PT. Such strains would be useful for vaccine production. In addition, knowledge of the mechanism of PT secretion might aid in the development of live-attenuated vaccines that secrete inactivated forms of PT, thus inducing a protective immune response.